INDIVIDUAL NATIONAL RESEARCH SERVICE AWARD APPLICATION FORM PHS 416
1. Effects of Antioxidant Vitamins on Autoimmune-Prone NZBxNZW F1 Mice
McCarthy, Brian A.
University of Medicine and Dentistry of New Jersey, Pathology Department
BMcC333@aol.com
4i. Native citizen of USA
5. 180 Biology
6. No Prior and/or Current NRSA Support (Individual or Institutional).
7a. Dates of Proposed Award: 9/7/98 - 9/7/00
7b. 12 months
8. Proposal not part of degree requirements.
Completed by Sponsor:
9. No Human Subjects
10. Vertebrate animals used
10a. IACUC Approval Date: _____________
10b. Animal Welfare Assurance No. ____________
11a. Sponsor's Name:
Raveche, Elizabeth S.
11b. University of Medicine and Dentistry of New Jersey
185 South Orange Avenue
Newark, NJ 07103
11c. Graduate School
11d. Pathology Department
12. Entity ID number: ___________________
14. Name of official in business office: ______________________
15. Applicant Certification and Acceptance.
I certify that the statements herein are true, complete and accurate to the best of my knowledge and I agree to comply with the Public Health Service terms and conditions if an award is issued as a result of this application. I am aware that any false, fictitious or fraudulent statements may subject me to criminal, civil or administrative penalties. I certify that I have read the National Service Award Service Assurance, that I will abide by the Assurance if an award is made, and that the award will not support residency training.
_______________________________________________Date:___________
16. Education: Jersey City State College BS Biology
UMDNJ Graduate School of Biomedical Sciences Ph.D. program
Goals. Autoimmune diseases may benefit from antioxidant treatment. Free radicals, molecules containing an unpaired electron, are known to pose a serious threat to systemic damage. Many nutrients are effective as quenchers of singlet oxygen in vitro, however protection against cardiovascular disease and cancer remains unproven. A method for in vivo analysis is proposed by adding to a diet of the basic essential antioxidants or subtracting from 32 antioxidants commonly sold in health food stores. New Zealand Black and White female mice are a murine model for autoimmune diseases like Systemic Lupus Erythmatosis (SLE). and will be used to test for pathology and longevity after antioxidant treatment.
Details. Antioxidant research is rapidly expanding and can have wide applications. A person with cancer doesn't think about vitamin therapy. Many in the medical establishment still insist taking vitamin supplements is a futile effort. The importance of serum levels of certain vitamins has been known to science since scurvy. Much more is now known about the exact organic reactions occurring with the various vitamins, but little has been done to quantify the effects and possible rewards of lifelong vitamin therapy or increased doses of certain vitamins in response to specific host challenges. The hazards of low levels of blood serum antioxidant levels were recently analyzed in a human study: Cases of both rheumatoid arthritis and chronic lymphocytic leukemia (CLL) had lower serum concentrations of vitamin E; alpha tocopherol and vitamin A; beta carotene and retinol in 1974 than their matched controls. These findings support those of a previous study that low antioxidant status is a risk factor for rheumatoid arthritis. They suggest a similar association for systemic lupus erythematosus (SLE) [1].
CLL, although not as severe as the acute form, is still a serious disorder and an aggressive level of research can potentially improve the health of many. CLL is the most frequent form of leukemia in western countries. The survival may be as short as a few months while others die from unrelated causes 25 years after diagnosis [2]. CLL appears to affect proportionally more women and can therefore be considered in the quest to study minority health concerns.
The Food and Agriculture Organization (FAO) of the United Nations compiles per capita consumption based on retail sales. This data goes back to 1961 and allows for long term evaluation of cancer risk and eating habits. The relationship between animal fat and two cancers, colorectal and breast, was shown without exception after comparing the extremes. Countries with the lowest intake such as Portugal, Greece, Spain, Romania and Bulgaria had lower rates than countries where intake was highest such as United Kingdom, Ireland, Belgium and Denmark.
However some countries like Iceland, which is one of seven countries with over 100 g/day animal fat intake, had comparable cancer rates with low fat consumers. In fact Icelanders are marginally safer than the Portugese, the lowest animal fat consumers in Europe. Icelanders consume twice as much fish (91 kg/yr in the 80's) as Europe's second biggest consumer, again the Portugese. Europe's third most prolific fish oil consumer, Finland, had even lower aggregate risk of breast and colorectal cancer than Iceland.[3]
The value of each antioxidant has largely been discovered independently. Linus Pauling helped create an entire vitamin C supplement industry by advocating its properties before congress and consuming a daily dose one hundred times the recommended daily allowance.[4] Beta carotene and alpha tocopherol have inhibited atherosclerotic lesions and reduced cholesterol in hypercholesterolemic rabbits.[5]
Vitamins A, C and E have generated the most antioxidant research but other agents have been studied for a positive effect on the antioxidant defense system and its related enzymes. Selenium, an important antioxidant that works synergistically with vitamin E has been studied as an anti-aging chemical. A human experiment tried to determine oxidative stress status and its significance in elderly subjects. Six parameters marking oxidative stress were evaluated in 52 elderly patients (mean age 85 +/- 6 years; range 74 to 98) admitted to a medium-term and long-term nursing home (n = 30) or a hospital ward (n = 22)were compared with those in 30 disease-free young subjects (age range 20-40 years). Plasma levels of thiobarbituric acid reactive substances vitamin E and selenium and activity of free-radical protective enzymes (erythrocyte superoxide dismutase, plasma and erythrocyte glutathione peroxidase) were assessed. Thiobarbituric acid reactive substances were higher and superoxide dismutase, erythrocyte and plasma glutathione peroxidase, and plasma selenium were lower in elderly patients than in young controls. There was no difference in vitamin E levels. In the nursing home population, erythrocyte superoxide dismutase was correlated with erythrocyte glutathione peroxidase and vitamin E, plasma glutathione peroxidase with erythrocyte glutathione peroxidase, vitamin E and selenium and erythrocyte glutathione peroxidase, vitamin E and selenium and erythrocyte glutathione peroxidase with vitamin E. Only the correlation between erythrocyte and plasma glutathione peroxidase was found in the hospitalized population. The team concluded: "Oxidative stress" assessed by six parameters was thus observed in the elderly population and could be considered as a "biological marker of aging". Supplementation with selenium or other anti-oxidants could be proposed.[6]
Superoxide has become one of the fastest growing areas of research. It is the most damaging form of free radical, and can be generated during the normal catyltic functions of enzymes and the oxidation of hemoglobin to methemoglobin. Fritz Haber produced free radicals from the reaction of superoxide and hydrogen peroxide in 1933 but Joseph Priestly was the first to hypothesize the dangers involved with an improper balance of oxygen. In 1775, while watching a candle burn faster in pure oxygen he surmised "as a candle...animal powers be too soon exhausted". Superoxide dismutase, discovered in 1969 by Joe M.McCord Ph.D and Irwin Fridovich catalyzes the elimination of superoxide. Since 1969 more than 22,000 scientific articles have been written on the subject. Three forms exist, in the mitochondria, cytosol and extracellular secretions. Many were surprised the body was capable of such a volatile reaction. When cells are diseased or injured, the normal metabolism of oxygen goes awry, leading to the increased production of superoxide. In addition, white blood cells intentionally produce superoxide in order to kill microorganisms. These white cells are activated by trauma and inflammation, in addition to infection. Hence, nearly all diseases involve the production of increased amounts of free radicals. Diseases of primary interest include heart attack, stroke, arthritis, cancer, AIDS, and neurological diseases such as ALS.[7]
(1) Specific Aims/ Hypothesis. Autoimmune-Prone NZBxNZW F1 Mice, an inbred line developed in the 1930's, are the murine model for autoimmunity and spontaneously develop autoimmune disease. An enhanced antioxidant system will delay the onset of autoimmune disease and increase life span.
(2) Background and Significance. Many diseases are treated from a symptoms first or what works approach and this can be a slippery slope in autoimmune disorders, since immunosuppression is a popular treatment. In particular, nucleoside analogs such as fludarabine and deoxycoformycin have had positive results, however immunosuppression usually produces unwanted side effects. The underlying pathology of autoimmune diseases is largely unknown but probably has genetic components. Dr. Raveche's UMDNJ pathology lab has demonstrated the deleterious effects of anemia on New Zealand Black mice. Young mice which had experimentally induced anemia had several characteristics in common with old autoimmune mice. Autoimmune NZB mice have a naturally occurring anemia due to the presence of anti-erythrocyte autoantibodies [8]. Some of the characteristic features of this mouse are hypergammagoblulinemia, autoantibodies, polyclonal B cell activation, CD5 B cell lymphocytosis in peritoneal cavity and or spleen, hyperdiploidy (aneuploidy), and a transplantable, splenic lymphoma [9].
Anemia is related to blood oxygen levels and it has been demonstrated that an inefficient antioxidant system has been linked to autoimmune disease. Antioxidant enzymes such as glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) as well as antioxidants nutrients, such as vitamin A, vitamin C, vitamin E and selenium, can participate in controlling lipid peroxidation. Because antioxidant enzymes play an important role in the protection against free radical damage, a decrease in the activities or expression of these enzymes may predispose tissues to free radical injury. The data, from studies with Menhaden fish oil, indicates that one of the mechanisms through which the n-3 lipids delay the onset of autoimmune diseases in NZBxNZW F1 (B/W) mice may be through maintenance of higher activities and expression of hepatic antioxidant enzymes. Menhaden Fish oil is highly purified and has been used as an n-3 lipid source in several experiments and clinical studies [10]. In this experiment the Menhaden Oil has been generously provided by Omega Proteins of Zapata Haynie (Reedville, Va.). J.R. Carlson Laboratories Inc. Arlington Heights, Il has generously provided the commonly available Norwegian Cod Liver Oil. These oils are to be used as a source of omega three polyunsaturated fatty acids (PUFA) eicosapentanoic acid (EPA) and docosahexanoic acid (DHA). One teaspoon of Cod Liver Oil provides 5000 IU of vitamin A, 500 IU vitamin D3 and 1 IU of Vitamin E. Each teaspoon also provides 1100 mg (22%) of Omega-3 fatty Acids composed of 500 mg (10 %) EPA, 550 mg DHA (11%) and 50 mg ALA (1%) DHA (Carlson Laboratories.).
The theory underlying the hypothesis is that autoimmune diseases are perhaps the end stage response of a weary immune system from free radical systemic damage. In a desperate attempt to prevent vital tissue damage, B cells proliferate clonally as targets. As numbers increase, some of these cells fall out of solution and grow as tumor cells. Therefore the maintenance of the proper structures involved in the antioxidant defense system is paramount to the prevention and treatment of autoimmune disease.
The idea that B-CLL is a condition of aging like hair loss or thymic involution suggests a difference in the labeling of the incidence and examination of the patient population.[11]
Natural prevention and treatment may provide a superior alternative to standard immunosuppressant treatment. Despite the degree of genetic predisposition to autoimmune diseases, an enhanced antioxidant defense system should improve mortality and survival rates. Modern western diets eat predominantly omega-6 essential fatty acids from vegetable oils, and too little omega-3. The Department of Health recommends doubling the amount of omega-3s we eat. Omega-3 essential fatty acids (EFAs) are derived mainly from fish oils. Omega-3s are believed to help reduce the risks of cardiovascular disease. Among the main materials required for fetal brain and CNS growth in late pregnancy are omega-3s (mainly DHA) and omega-6 EFAs (mainly arachidonic acid [AA]). These come from the mother's diet. Several formulae for preterm infants now contain DHA and AA to aid optimum brain, nerve and retinal development. One manufacturer has begun to include DHA and AA in formulae for term babies. Breast milk contains DHA and AA, derived from the mother's diet. Eating oily fish in pregnancy has been found to have a slight beneficial effect on birth weight and length of gestation. Eating fresh or canned oil-rich fish (e.g. tuna, kippers, herring, mackerel, salmon, sardine, pilchards,) twice or three times a week can be encouraged as part of a healthy balanced diet, in pregnancy and for all the family. As well as containing omega-3 polyunsaturates, oily fish is a good source of protein and vitamins A and D. Alternatively, a fish-oil supplement may be taken. Cod liver oil is best avoided during pregnancy, because of concerns over the possible teratogenicity of vitamin A.[12]
The molecular lesions of human familial and common B-CLL remain unknown [9]. A growing body of epidemiological and experimental evidence indicates there is an inverse relationship between the risk of cancer at several sites and the consumption of foods rich in vitamins A and C. The involvement of vitamin A in the control of growth, differentiation and function of epithelial tissue and that of vitamin C in the maintenance of tissue integrity and host defense provides a logical base for examining their relationship to cancer. Both vitamins studied were effective in delaying the appearance of the disease in the case of Schwartz Lymphoblastic Leukemia in challenged mice [13]. NZBxNZW F1 mice in groups of fifteen were fed a diet of either (n-3 rich) menhaden fish oil, Krill oil or (n-6 rich) Corn oil with equal amounts of vitamin E. After 11 months the control group had no survivors, the krill oil group had 3 survivors and the menhaden group had 7 survivors; a survival percentage of 46.7 compared to zero [10].
White Rabbits were made hypercholesterolemic with an atherogenic diet of .5% cholesterol, 3% peanut oil and 3% coconut oil for eight weeks. They were then treated with beta carotene and alpha tocopherol. Combination of both antioxidants significantly decreased total and LDL cholesterol concentrations, susceptibility of LDL to oxidation ex vivo as well as atherosclerotic lesion area and intimal thickness at aortic arch and thoracic aorta [5].
Vitamin E, RRR-alpha-tocopherol plays an essential role in protecting cell membrane PUFA against oxidation (Tappel 1962). Kagan and co-workers (1990) have shown protection against the damaging effects of phospholipase A2 and its products (i.e. free fatty acids and lysophospholipids). These actions could be particularly important in immune cells in which arachidonic acid and phospholipase A2 are integral parts of eicosanoid biosynthesis (Chilton et al. 1996). Increased dietary vitamin A has been shown to antagonize vitamin E in poultry (Abawi and Sullivan 1989, Tengerdy and Brown 1977) and the inclusion of Cod Liver oil (CLO) to precipitate vitamin E deficiency has historically been a common practice among researchers.[14] All mice in this experiment will receive additional vitamin E and CLO is included as an historical reference. Any difference in the Menhaden Fish oil group and the CLO group may be due to this important difference.
Cod Liver Oil has been used as an historical marker in many oil experiments due to the large number of previous experiments with consistent results and the lengthy history of CLO. By the 1840's there were many accounts of CLO's medicinal value;"Whether the disease of rickets be in its most severe form, with swollen joints and crooked legs, or at its commencement, the cod liver oil will supersede every other means of cure" and "A cause of inflammation of the eyelids and intolerance of light was cured in ten days with cod liver oil as were several cases of conjunctivitis of the cornea.". De Jongh (1849) speculated about the mode of action of various cod liver oils in relation to their fatty acid make-up.[15] Although the American Heart Association Report of 1957 was only willing to venture a qualified stance "The fat content and total calories in the diet are probably important factors."(Page et al. 1957)[16], the relationship between the proper balance of dietary fat and a functional immune system was recognized early by some: "Pastries made with butter, are little better than poison for children, but the fond mother treats her darling boy with these dainties and then wonders how he got the colic" (Buchan 1813).[15]
In 1842, The Lancet contained two articles on the treatment and prevention of scurvy. In the 1930's Szent-Gyorgi isolated ascorbic acid, which he called hexuronic acid. He isolated several other important compounds of the vitamin C complex, which have been largely ignored. Szent-Gyorgi tried on several occasions to cure scurvy with ascorbic acid but was reportedly unsuccessful. Eventually Szent-Gyorgi discovered a bioflavonoid compound from lemons he called citrin or vitamin P. Other components of the C complex include vitamin K, which supports blood clotting mechanisms; the J factors, that enhance oxygen carrying by the blood, some polyphenols, catechin, enzymes and tyrosinase, a copper-activating enzyme that aids in the formation of hemoglobin.[17]
Almost all animals produce their own vitamin C (ascorbic acid). The guinea pig and the monkey resemble man in requiring exogenous vitamin C [4]. Dietary ascorbic acid (AA) improves the activity of macrophages and the ex vivo response to mitogens in fish which lack endogenous AA. Vitamin C deficient pigs which are unable to synthesize ascorbic acid were challenged with mitogens concanavalin A, pokeweed mitogen (PWM) lipopolysaccharide (LPS) and phytohemmagutin. The data indicated that ascorbic acid selectively influences the proliferation of B lymphocytes [18].
One of the most potent defense mechanisms of the body is the destruction of invading bacterial cells by the leukocytes of the blood (phagocytosis). It has been known for over fifty years that vitamin C is needed for effective phagocytic activity of leukocytes. It is also known that stresses lead to a decrease in the leukocyte concentration below the phagocytically effective level [4]. Optimum intake of antioxidant vitamins may well exceed the recommended daily value.
CLL has been theorized to be a dual problem of clearance, involving apoptosis, programmed cell death and proliferation increasing the chances of tumor formation. Ascorbic Acid (vitamin C, VC) seems to play a dual role involving both of these extremes. Ascorbic Acid was initially evaluated for its ability to prevent cancer because of its antioxidant properties (Cameron et al., 1979; Pavelic et al 1989). However the result of in vitro studies demonstrate that VC exhibits selective toxicity towards malignant melanoma cells (Bram et al.,1980), human leukemia cells (Park et al., 1980) neuroblastoma cells (Prasad et al. 1979), tumor ascites cells (Liotti et al., 1984) and other malignant cell lines (Leung et al., 1993) with VC acting as a pro-oxidant and not an antioxidant (De Laurenzi et al., 1995) The mechanism responsible for the antitumor activity of VC appears to be related to the level of L-ascorbate and its oxidative product, dehydoascorbate, which generates intracellular H2O2 and initiates membrane lipid peroxidation. Subsequently, hydroxyl radicals may be generated via a Fenton-type reaction and produce DNA breaks and protein alterations. Tumor cells appear to be more susceptible to these oxidative stresses than their non-malignant counterparts because of the altered patterns of antioxidant enzymes in the tumor cells (Taper et al., 1987; Leung et al., 1993; De Laurenzia et al., 1995). The results of a recent study by De Laurenzi et al,. and co-workers (1995) demonstrated that physiological levels of ascorbic acid were cytotoxic to neuroectodermal cancer cells. The author suggests that recycling of ascorbate by elevated levels of NADH dependent semihydroascorbate reductase enhanced the pro-oxidant action of ascorbic acid which led to a pro-oxidant activation of programmed cell death [19].
Krumdieck and Butterworth in their discussion of the pathogenesis of atherosclerosis conclude that "vitamin C seems to occupy a position of unique importance by virtue of its involvement in two systems; the maintenance of vascular integrity and the metabolism of cholesterol to bile acids," It strengthens the blood vessels through its participation in the synthesis of connective tissue [4].
Vitamin K is normally present in the human diet. Vitamin K is a generic term for compounds that include phytonadione(VK1), the menaquinone series (VK2) and menadione (VK3). Physiologically the natural K vitamins VK1 and VK2 are known to act as cofactors for gamma-carboxylation of selected glutamates at the n-termini of prothrombin and other VK-dependent coagulation factors. Geranylgeraniol, a polyprenylalcohol composing the side chain of VK2 was previously reported to be a potent inducer of apoptosis in tumor cell lines[18]. VK2 and all-trans retinoic acid enhanced apoptosis of leukemia cells in vitro, in particular the combination of ATRA 100 nM and MK5 at 10 uM. This combination appears to be achievable in vivo using an intravenous injection of 30 mg (67.6 Mm), the normal clinical dose of MK4, however more detailed studies will be required regarding the toxicity of extended exposure to supra-physiological concentrations of Mks [21].
The rationale for the inclusion of vitamin K3 in a 100 to 1 ratio with vitamin C is included in the Jamison et al., Tissue and Cell article (1996): Vitamin K3 (2-methyl-1,4-naphthoquinone) was initially evaluated for its antitumor activity because it possesses the quinone moiety common to several known antitumor agents (e.g. adriamycin and mitoxantrone) and because it does not induce the cardiotoxcicity observed in these other antitumor agents (Nutter et al,. 1991). Vitamin K3, a synthetic derivative of vitamin K1, exhibits in vitro antitumor activity against leukemias and lymphomas as well as against cancers of the liver, cervix, nasopharynx, colon, lung, stomach, and breast (Nutter et al,. 1991; Wu et al,. 1993a,b), Furthermore, VK3 has been shown to be equally potent against human multiple drug resistant and drug sensitive cells, as well as against human tumor explants from patients with neoplasms which are refractory to other types of chemotherapy (Nutter et al, 1991). VK3 has been shown to induce apoptotic-like cell death in hepatocytes and a variety of tumor cell lines (McConkey et al,. 1988; Juan & Wu, 1993; Wu et al,. 1993a). Taper and his associates demonstrated that when VC and VK3 were combined in a ratio of 100:1, the combination exhibited tumor specific antitumor activity with exposure times as short as one hour and at doses 10-50 times lower than when either vitamin was administered alone (Noto et al,. 1989) [19].
The extract of Gingko biloba referred to as EGb 761 is one of the most popular plant extracts sold in Europe use to alleviate symptoms associated with a range of cognitive disorders It has recently been approved in Europe for treatment of dementia. The mechanism in the central nervous system is only partially understood but the main effects seem to be related to its antioxidant properties, which require the synergistic action of the flavonoids, the terpenoids (ginkgoloids, bilobalide) and the organic acids, principle constituents of EGb. They act as scavengers for free radicals which have been considered the mediators of the excessive lipid peroxidation and cell damage observed in Alzheimer disease. A double blind randomized human trial concluded that the compounds in EGb acted synergistically on diverse processes involved in homeostasis of inflammation and oxidative stress.[22]
Studies of lipid metabolism have suggested that oxidative modifications of low density lipoprotein (LDL) accelerate atherogenesis and this led to claims of vitamin E's ability to reduce incidence of nonfatal myocardial infarction, which were unsubstantiated by further research. Proposed methods include stimulation of monocyte and platelet adhesion to the endothelium, inhibition of vasodilation, stimulation of synthesis of autoantibodies, and proliferation of smooth muscle cells leading to the promotion of foam cells and fatty streaks in the arterial intima. Natural antioxidants present in the diet may inhibit the oxidative modification of LDL and slow the progression of atherosclerosis. Lycopene has also experienced initial success in a 10 country European EURAMIC study comparing the carotenoids alpha-carotene, beta-carotene and Lycopene. Supplementation has not yet proven effective in studies so focus has turned to other components of vegetables. Beta carotene is found in relatively high concentrations in carrots and pumpkins as lycopene is found in tomatoes, guava and watermelon. The relationship between polyunsaturated fats and carotenoids showed the protective level of lycopene increased at each increasing level of polyunsaturated fats and was significant in individuals whose adipose tissue contained more than 16.1 percent of the fat in the form of polyunsaturates. The EURAMIC study suggests dietary beta carotene plays a protective role for smokers. The team concluded "Lycopene or some substance highly correlated which is in a common food source, may contribute to the protective effect of vegetable consumption on myocardial infarction risk. [23]
Inspec Fine Chemicals has generously donated Butylated Hydroxy Toluene which markets under the brand Ionol CP and is well known as the food preservative BHT. Ionol is a strong non toxic, non-staining multipurpose antioxidant used in food, polymers like rubber, polyurethanes and lubricants among others.
Ionol was first used in cardiac research with rats at Moscow State University to show lipid peroxidation was a decisive link in the pathogenesis of stress damage to the heart. Ionol completely prevented activation of lipid peroxidation, which develops normally in the myocardium under stress, eliminating stress damage to the heart. It was concluded that Ionol prevents hypoxic damage to the heart and significantly increases the rate and degree of posthypoxic recovery of contractile function of the heart upon reoxygenation.[24] The amount of Ionol used was based on amounts used in a toxicoligal evaluation by the World Health Organization. Amounts of 0.25% and 0.5% by dry weight were fed to 60 FAF male mice. The mean life span of the test animals was significantly greater than controls, being 17.0 +/-5.0 and 20.9+/-4.7 months respectively as compared to 14.5+/- 4.6 months.[25].
The UMDNJ Pathology Department Basic Antioxidant Test diet (PDBAT)
provides the basis for additional testing through addition of other substances. The UMDNJ Pathology Department Broad Range Antioxidant Test diet (PDBRAT) provides a basis for testing through deletion of substances..
(3) Research Design and Methods. NZBxNZW F1 mice from Jackson Laboratories (Bar Harbor, Me) will be randomly split into four groups: All diets will be based on AIN93 The widely used Cod Liver Oil will be used as the control group at 20%. All diets, test and control, will be supplemented with the 7 basic antioxidants: A (retinol and beta-carotene), B12 ,C,E, K,and selenium. Three test groups will be given purified Menhaden fish oil.(20%). Group One will be given only the basic antioxidants. Group two will be given a broader range of 25 additional antioxidants including Coenzyme Q, Super Oxide Dismutase and Lycopene. Group three will be treated with the basic antioxidants and the addition of butylated hydroxy toluene (Ionol) in the amount of 3300 ppm.
10 kg each of all diets
Diet one: UMDNJ Pathology Department Cod Liver Antioxidant Test Diet (PDCAT): AIN-93 + 20% Cod Liver Oil + 7 basic antioxidants (increase in selenium, Retinol, B12 and the addition of D-Alpha tocopherol, all trans-beta carotene, vitamin K3 and vitamin C.
Vitamin Mixture: (20 g/kg) supplied per kg diet, in mg: (In addition to PMI 5755, separate from CL Oil) to contain at least:
Retinol: 5 mg/kg
all trans-beta-carotene, 70 mg/kg;
vitamin B12 (Cobalamin) 0.005 mg/kg;
L ascorbic acid as vitamin C complex: (9 to 1) 700 mg/kg;
d-alpha-tocopherol acetate: 300 mg/kg;
vitamin K3 (2-methyl-1,4,napthaquinone): 7 mg/kg;
Selenium (L-Selenomethionone): 0.4 mg/kg; (2x PMI5755)
Analyte Cost
Vitamin A (carotenes) $75
Vitamin B1 $50
Vitamin B2 $50
Vitamin B3 $50
Vitamin B5 $68
Vitamin B6 $70
Selenium $51
Zinc $18.25
Manganese $18.25
Sample Preparation $5
We would like to have 250 grams or more of sample if that much is available.
ralston.com
Diet two: UMDNJ Pathology Department Basic Antioxidant Test Diet (PDBAT): AIN-93 + 20% Mehanden Fish Oil + 7 basic antioxidants (increase in selenium, Retinol, B12 and the addition of D-Alpha tocopherol, all trans-beta carotene, vitamin K3 and vitamin C.
Vitamin Mixture: (20 g/kg) supplied per kg diet, in mg: (In addition to PMI 5755, separate from CL Oil) to contain at least:
Retinol: 5 mg/kg
all trans-beta-carotene, 70 mg/kg;
vitamin B12 (Cobalamin) 0.005 mg/kg;
L ascorbic acid as vitamin C complex: (9 to 1) 700 mg/kg;
d-alpha-tocopherol acetate: 300 mg/kg;
vitamin K3 (2-methyl-1,4,napthaquinone): 7 mg/kg;
Selenium (L-Selenomethionone): 0.4 mg/kg; (2x PMI5755)
Diet three: UMDNJ Pathology Department Broad Range Antioxidant Test Diet (PDBRAT): AIN-93 + 20% Menhaden Fish Oil + 7 basic antioxidants (increase in
selenium, Retinol, B12 and the addition of D-Alpha tocopherol, all trans-beta
carotene, vitamin K3 and vitamin C. Plus a broad range antioxidant complex.
Vitamin Mixture (20 g/kg) supplied per kg diet, in mg: (In addition to AIN93, separate from fish Oil) to contain at least: the 7 antioxidants included in PDBAT (*):
*Retinol: 5 mg/kg
*all trans-beta-carotene, 70 mg/kg;
*vitamin B12 (Cobalamin) 0.005 mg/kg;
*L ascorbic acid as vitamin C complex: (9 to 1) 700 mg/kg;
*d-alpha-tocopherol acetate 300 mg/kg;
*vitamin K3 (2-methyl-1,4,napthaquinone): 7 mg/kg;
*Selenium (L-Selenomethionone): 0.4 mg/kg; (2x PMI5755)
Plus additional Broad Range Antioxidants: Dose of 2 pills per Kilogram
20 pills of 100 ct multi VS 1175 =$14.66
vitamin A (Beta Carotene and mixed carotenoids 20,000 IU/kg
vitamin B1 (Thiamine) 50 mg/kg
vitamin B2 (Riboflavin) 50 mg/kg
vitamin B3 (Niacinamide) 50 mg/kg
vitamin B5 (Pantothenic acid) 50 mg/kg
vitamin B6 (Pyridoxine) 50 mg/kg
N-acetyl Cysteine (glutathione precursor) 100 mg/kg
Glutathione 100 mg/kg
Selenium (L-Selenomethionone): 0.15 mg/kg;
Zinc (Picolinate) 30 mg/kg
Green tea extract (30% polyphenols) 50 mg/kg
Coenzyme Q-10 20 mg/kg
Quercitin (Bioflavonoid) 50 mg/kg
Alpha Lipoic acid(Thioctic acid) 20 mg/kg
Garlic (Odorless) 200 mg/kg
Grape seed extract (min 85% proanthocyanids) 20 mg/kg
Manganese (Gluconate/Citrate) 30 mg/kg
20 pills 60 count Carotenoids VS-1271 =$11.21
Lycopene (carotenoid) 10 mg/kg
Alpha Carotene 2.5 mg/kg
Beta Carotene 7.5 mg/kg
Phytofluene 0.066 mg/kg
Leutin (Marigold flower) 10 mg/kg
Zeaxanthin (Marigold flower) 0.52 mg/kg
Phytoene 0.196 mg/kg
20 pills of Rutin KA-1335 =$3.99
Rutin (Bioflavonoid) 500 mg/kg
20 pills SOD VS-1038 =$5.56
Super Oxide Dismutase 0.25 mg/kg
20 pills Ginkgo VS-1134 =$9.57
Ginkgo Biloba (EGB 761; 24% ginkgoheterosides) 120 mg/kg
total retail cost = $44.24
+ $4.25 shipping = $48.99
Diet four: Pathology Department Basic Antioxidants plus Ionol (PDBAI): Basic test diet (PDBAT) AIN-93 + 20% Menhaden Oil + 7 basic antioxidants (increase in selenium, Retinol, B12 and the addition of D-Alpha tocopherol, all trans-beta carotene, vitamin K3 and vitamin C. Plus Ionol, (BHT) administered at 3300 ppm dry weight or 0.33% of dry weight. of 10 kilograms totals 33 grams of Ionol.
Total feed order = 10 kg PDCAT, 10 kg PDBAT, 10 kg PDBRAT and 10 kg PDBAI
Oil Sources: Cod Liver Oil courtesy of J.R. Carlson Laboratories Inc. Arlington Heights, Il
Menhaden Fish Oil courtesy of Omega Protein, Zapata Haynie (Reedville, Va.).
Vitamin Source :The Vitamin Shoppe 4700 West Side Ave, North Bergen, NJ 07047 (800-223-1216)
Ionol Source: Inspec Fine Chemicals
Fresh food provided daily in the evening, precautions to prevent oxidation of lipids and diet. Mice will be maintained in plastic cages under 12 hour light dark cycles in the UMDNJ animal facility. Body weights and proteinuria taken once a month and NIH Guidelines strictly followed. Upon death the following will be performed on all mice: complete autopsies including CBC. spleen weight, peritoneal cell count, absolute lymphocyte counts, spleen immunophenotyped and cell cycle analysis, assays of antioxidant vitamin levels. Serum samples will be analyzed for carotenoids, retinol and alpha tocopherol by high performance liquid chromatography assays. Carotenoids to be quantified will include alpha carotene, beta carotene, beta cryptoxanthin and leutin plus zeaxanthin. Vitamin C will be assayed by measuring the reduction of 2,6-dichlorophenolindophenol by ascorbic acid present in deproteinized serum or tissue homogenate. The change in absorbance (at 520nm) due to reduction by vitamin C in the sample is calculated by the equation (RB-RBb)-(S-Sb) where RB is the reading
of the reagent blank, RBb is the reagent blank after reduction. S is the sample reading and Sb is the sample after reduction. Pure L-ascorbic acid will be used for preparation of the standard curve [12]
Item 29d. Selection of Sponsor and Institution.
(1) Explain why the sponsor and institution were selected to accomplish the research training goals.
Dr. Raveche's pathology lab has performed many innovative experiments involving autoimmune disorders. These include a study relating the adverse effects of anemia upon autoimmune mice. This experiment seeks to build on that knowledge by examining the potential positive effects of a fortified diet
References and Notes
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